Mutants of green fluorescent protein

ABSTRACT

The present invention provides mutants of the Green Fluorescent Protein (GFP) of  Aequorea victoria . Specifically provided by the present invention are nucleic acid molecules encoding mutant GFPs, the mutant GFPs encoded by these nucleic acid molecules, vectors and host cells comprising these nucleic acid molecules, and kits comprising one or more of the above as components. The invention also provides methods for producing these mutant GFPs. The fluorescence of these mutants is observable using fluorescein optics, making the mutant proteins of the present invention available for use in techniques such as fluorescence microscopy and flow cytometry using standard FITC filter sets. In addition, certain of these mutant proteins fluoresce when illuminated by white light, particularly when expressed at high levels in prokaryotic or eukaryotic host cells or when present in solution or in purified form at high concentrations. The mutant GFP sequences and peptides of the present invention are useful in the detection of transfection, in fluorescent labeling of proteins, in construction of fusion proteins allowing examination of intracellular protein expression, biochemistry and trafficking, and in other applications requiring the use of reporter genes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/668,168, filed Sep. 24, 2003, which is a continuation of U.S. patentapplication Ser. No. 09/472,065, filed Dec. 23, 1999, now U.S. Pat. No.6,638,732, which is a continuation of U.S. patent application Ser. No.08/970,762, filed Nov. 14, 1997 (now abandoned), which claims thebenefit of U.S. Provisional Application No. 60/030,935, filed Nov. 15,1996, the disclosures of which applications are entirely incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is in the fields of molecular and cellular biology. Moreparticularly, the invention is directed to mutants of the genes encodingGreen Fluorescent Protein (GFP) and the proteins encoded by thesemutants. The mutant GFPs are used to allow detection of eukaryotic andprokaryotic cells transfected or transformed with extrinsic genes, andto label proteins of interest to facilitate their localization withinviable cells.

2. Related Art

Transfection of Foreign Genes

To study the function of a gene, a technique that is commonly employedis the transfer of the gene into a new cellular environment. Thisprocess, called “transfection,” provides several advantages to thegenetic scientist. For example, the cellular protein encoded by the genecan often be more easily studied by transferring the gene into a cell ororganism that normally does not produce the protein, and then examiningthe effect of this protein on the host cell. The existence and functionof regulatory genetic sequences (e.g., promoters, inhibitors andenhancers) may be elucidated by transfection of foreign genes into cellscontaining the regulatory sequences. The transfer of non-native oraltered genes into a host cell also allows for large-scale production ofthe proteins encoded by the genes, a process upon which much of thecurrent biotechnology industry is based. Transfection of plant embryoswith foreign genes has provided genetically engineered plants that aremore resistant to adverse environmental conditions or that are morenutritionally rich. Finally, gene transfer methods allow theintroduction of new or mutated genes into whole organisms. This lattercapability provides the opportunity for the construction of stablemodels of mammalian diseases, for large-scale production of proteins inthe milk of transgenic lactating animals, and for the possibility ofgenetic therapy for certain diseases.

A variety of techniques has been used to transfect non-native genes intocells (reviewed in Sambrook, J., et al., Molecular Cloning, a LaboratoryManual, 2nd Ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress, pp. 16.30-16.55 (1989); Watson, J. D., et al., Recombinant DNA,2nd Ed., New York: W.H. Freeman and Co., pp. 213-234 (1992)). Thesetechniques include biological methods such as the use of viruses (e.g.,adenovirus or certain retroviruses for mammalian cells, baculovirus forinsect cells and bacteriophages for bacterial cells) or bacteria (e.g.,Agrobacterium for plant cells), chemical methods such as calciumphosphate precipitation, DEAE-dextran-mediated endocytosis orliposome-mediated transfection, and physical methods such aselectroporation or direct microinjection. For transfection of mammaliancells, the techniques most commonly employed currently arevirus-mediated transfection, lipofection and electroporation.

Detection of Gene Transfer

Regardless of the method used, however, simply attempting to transfect acell does not guarantee that a majority (or even any) of the targetcells will take up and/or express the exogenous DNA. Indeed, it has beensuggested that the success rate of even the most optimal techniques usedfor transfection results in stable transfer of exogenous DNA is far lessthan 1% (Watson, J. D., et al., Recombinant DNA, 2nd Ed., New York: W.H.Freeman and Co., pp. 216, 218 (1992)). Thus, it is usually critical todetermine which target cells have received and/or incorporated thegene(s) being transfected, for which a number of methodologies have beenused.

Expression

The most obvious of these methods is to simply examine the target cellsfor expression of the exogenous gene. In this method, the transfectedcells are grown in vitro and assayed for the presence of the proteinencoded by the transferred gene. These assays are usually accomplishedusing immunological techniques such as Western blotting, ELISA or RIA.This type of technique is only useful, however, if the protein isproduced in relatively high amounts (generally at the microgram level orabove) and if suitable antibodies are available, neither of which is thecase for some transfected genes.

In those cases where protein expression cannot be examined,incorporation of exogenous genes can be determined by assaying thetarget cells for production of the mRNAs corresponding to thetransferred genes. One very common technique for this determination isNorthern blotting (Alwine, J. C., et al., Proc. Natl. Acad. Sci. USA74:5350-5354, 1977), in which RNA molecules are isolated from cells,separated by gel electrophoresis and electroblotted onto a solid support(e.g., nitrocellulose or nylon). The solid support is then overlaid withradiolabelled cDNAs corresponding to the transfected gene, whichhybridize on the solid support to their complementary mRNAs. Afterexposing the blot to photographic film, the samples containing theexpressed transgene are easily determined. While this method is moresensitive than those directly measuring protein expression, Northernblotting still relies on actual expression of the gene by the targetcells, which is not always the case.

Selection

Another method for determining gene transfer, alternative to directlymeasuring gene expression, is to examine the effect of the gene on thetransfected cells. For example, some transfected genes will confer upontheir host cells the ability to grow in selective culture media or undersome other environmental stress which non-transfected cells cannottolerate. Genes of interest are often engineered into sequencesconferring, for example, antibiotic resistance upon the recipient cells.Transfectants with these constructs will thus carry not only the gene ofinterest but also the antibiotic resistance gene which allows them togrow in antibiotic-containing media. Since non-transfected cells willnot possess this resistance, any cell able to grow in media containingantibiotic will contain the resistance marker (the so-called “selectablemarker”) and the transgene that is linked to it. Selectable markerscommonly used in such an approach are the neomycin (neo), ampicillin(amp) and hygromycin (hyg) resistance genes.

In the same way, selectable markers conferring on the transfected cellsa metabolic advantage (e.g., ability to grow in nutrient-deficientmedia) have been used successfully. Examples of these types ofselectable markers include thymidine kinase (Bacchetti, S., and Graham,F. L., Proc. Natl. Acad. Sci. USA 74:1590-1594 (1977); Wigler, M., etal., Cell 11:223-232 (1977)) and xanthine-guaninephosphoribosyltransferase (Mulligan, R. C., and Berg, P., Proc. Natl.Acad. Sci. USA 78:2072-2076 (1981)), which impart to their recipientsthe ability to grow, using metabolic rescue pathways encoded by themarker genes, in media that inhibit vital metabolic pathways innon-transfected cells. Again, any cells able to grow in such media willcontain the transgene linked to the marker gene.

Selection methods such as these often require weeks of culturing of thecells, continuously under selective pressure, to provide a relativelypure population of stable transfectants. Many uses of transfected cells,however, are conducted within hours of transfection, far too soon todetermine transfection success using either the expression or selectionmethods described above. These types of applications are facilitated bya third approach—the use of “reporter genes”.

Reporter Genes

Reporter genes are analogous to selectable markers in that they areco-transfected into recipient cells with the gene of interest, andprovide a means by which transfection success may be determined. Unlikeselectable markers, however, reporter genes typically do not confer anyparticular advantage to the recipient cell. Instead reporter genes, astheir name implies, indicate to the observer (via some phenotypicactivity) which cells have incorporated the reporter gene and thus thegene of interest to which it is linked. A number of reporter genes havebeen used, including those operating by biochemical or fluorescentmechanisms, each with its own advantages and limitations.

Biochemical Reporter Genes

Some commonly used reporter genes encode enzymes or other biochemicalmarkers which, when active in the transfected cells, cause some visiblechange in the cells or their environment upon addition of theappropriate substrate. Two examples of this type of reporter sequenceare the E. coli genes lacZ (encoding β-galactosidase or “β-gal”) andgusA or iudA (encoding β-glucuronidase or “β-glu”); the former is oftenused as a reporter gene in animal cells (Hall, C. V., et al., J. Mol.Appl. Genet 2:101-109 (1983); Cui, C., et al., Trangenic Res. 3:182-194(1994)), the latter in plant cells (Jefferson, R. A., Nature 342:837-838(1989); Watson, J. D., et al., Recombinant DNA, 2nd Ed., New York: W.H.Freeman and Co., pp. 281-282 (1992); Hull, G. A., and Devic, M., Meth.Mol. Biol. 49:125-141 (1995)). These bacterial sequences are useful asreporter genes because the recipient cells, prior to transfection,express extremely low levels (if any) of the enzyme encoded by thereporter gene. When transfected cells expressing the reporter gene areincubated with an appropriate substrate (e.g., X-gal for β-gal or X-glucfor β-glu), a colored or fluorescent product is formed which can bedetected and quantitated histochemically or fluorimetrically.

Another often-used reporter gene is the bacterial gene encodingchloramphenicol acetyltransferase (CAT), which catalyzes the addition ofacetyl groups to the antibiotic chloramphenicol (Gorman, C. M., et al.,Mol. Cell. Biol. 2:1044-1051 (1982); Neumann, J. R., et al.,BioTechniques 5:444-446 (1987); Eastman, A., BioTechniques 5:730-732(1987); Felgner, P. L., et al., Ann. N.Y. Acad. Sci. 772:126-139(1995)). After transfection, recipient cells are lysed and the lysatesare incubated with radiolabelled chloramphenicol and an acetyl donorsuch as acetyl-CoA, or with unlabeled chloramphenicol and radiolabeledacetyl-CoA (Sleigh, M. J., Anal. Biochem. 156:251-256 (1986)). Ifexpressed in the cells, CAT transfers acetyl groups to chloramphenicol,which is then easily assayed by chromatographic techniques, therebygiving an indication of the incorporation of the co-transfected gene ofinterest by the recipient cells.

Using reporter genes in this way, populations of cells, or even singlecells, can be rapidly assayed for their incorporation of the exogenousgene linked to the reporter gene. Since they do not rely directly on theexpression of the gene of interest, assays of transfection success usingreporter genes are usually simpler and more sensitive than thosemeasuring mRNA or protein production from the transgene (Watson, J. D.,et al., Recombinant DNA, 2nd Ed., New York: W.H. Freeman and Co., p. 155(1992)). However, the use of reporter genes is severely limited in thatit usually requires sacrifice (fixation) of the cells prior to assay,and therefore cannot be used for assaying living cells or cultures.Thus, alternative means for determining the incorporation of thetransgene in viable cells have been developed.

Fluorescent Reporter Genes

An example of viable reporter genes that are rapidly gaining widespreaduse are those that are fluorescence-based. These genes encode proteinswhich are either naturally fluorescent or which convert a substrate fromnonfluorescent to fluorescent. Assays using this type of reporter geneare non-destructive and, owing to the availability of sophisticatedfluorescence detection systems, are often more sensitive thanbiochemical reporter gene assays.

One example of a fluorescence reporter gene is the luciferin-luciferasesystem (Bronstein, I., et al., Anal. Biochem. 219:169-181 (1994)). Thissystem utilizes the gene for luciferase, an ATPase enzyme isolated fromfireflies (Gould, S. J., and Subramani, S., Anal. Biochem. 175:5-13(1988)) and other beetles (Wood, K. V., et al., J. Biolumin. Chemilumin.4:289-301 (1989)), or from certain bioluminescent bacteria (Stewart, G.S., and Williams, P., J. Gen. Microbiol. 138:1289-1300 (1992);Langridge, W., et al., J. Biolumin. Chemilumin. 9:185-200 (1994)). Foruse as a reporter gene, the luciferase gene is placed into a vector alsocontaining the gene of interest, or separate vectors containing theluciferase gene and the gene of interest are mixed together. Cells arethen transfected with the vector(s) and treated with the luciferasesubstrate luciferin which is rendered luminescent (and impermeant)intracellularly by the action of the luciferase. Cells containing theluciferase gene, and thus the gene of interest linked to it, can then berapidly and sensitively observed using luminescence detectors such asluminometers.

To provide a further increase in sensitivity, attempts have been made touse genes from certain cyanobacteria which encode naturally fluorescentphycobiliproteins such as phycoerythrin and phycocyanin. These proteinsare among the most highly fluorescent known (Oi, V. T., et al., J. CellBiol. 93:981-986 (1982)), and systems have been developed that are ableto detect the fluorescence emitted from as little as onephycobiliprotein molecule (Peck, K., et al., Proc. Natl. Acad. Sci. USA86:4087-4091 (1989)). Phycobiliproteins also have the advantage of beingnaturally fluorescent, thus eliminating the time-consuming steps of theaddition of exogenous substrates for their detection as is required forluciferase and biochemical reporter genes. However, thephycobiliproteins have proven extremely difficult to engineer into geneconstructs in such a way as to maintain their fluorescence (Heim, R., etal., Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994)), and thus are notcommonly used as reporter genes in assaying the transfection ofmammalian cells.

Thus, the ideal reporter gene would encode a naturally fluorescentprotein (for ease of use following transfection) that is highlyfluorescent (for increased sensitivity) and easily engineered (formaintenance of fluorescence). Such a system has recently been developed,using the Green Fluorescent Proteins (GFPs) isolated from certain marinecnidarians.

GFP

Overview

GFPs are involved in bioluminescence in a variety of marineinvertebrates, including jellyfish such as Aequorea spp. (Morise, H., etal., Biochemistry 13:2656-2662 (1974); Prendergast, F. G., and Mann, K.G., Biochemistry 17:3448-3453 (1978); Ward, W. W., Photochem. Photobiol.Rev. 4:1-57 (1979) and the sea pansy Renilla reniformis (Ward, W. W.,and Cormier, M. J., Photochem. Photobiol. 27:389-396 (1978); Ward, W.W., et al., Photochem. Photobiol. 31:611-615 (1980)). The GFP isolatedfrom Aequorea victoria has been cloned and the primary amino acidstructure has been deduced (FIG. 2; Prasher, D. C., et al., Gene111:229-233 (1992)) (SEQ ID NOs:3, 4). The chromophore of A. victoriaGFP is a hexapeptide composed of amino acid residues 64-69 in which theamino acids at positions 65-67 (serine, tyrosine and glycine) form aheterocyclic ring (Prasher, D. C., et al., Gene 111:229-233 (1992);Cody, C. W., et al., Biochemistry 32:1212-1218 (1993)). Resolution ofthe crystal structure of GFP has shown that the chromophore is containedin a central α-helical region surrounded by an 11-stranded β-barrel(Ormö, M., et al., Science 273:1392-1395 (1996); Yang, F., et al.,Nature Biotech. 14:1246-1251 (1996)). Upon purification, native GFPdemonstrates an absorption maximum at 395 nanometers (nm) and anemission maximum at 509 nm (Morise, H., et al., Biochemistry13:2656-2662 (1974);Ward, W. W., et al., Photochem. Photobiol.31:611-615 (1980)) with exceptionally stable and virtuallynon-photobleaching fluorescence (Chalfie, M., et al., Science263:802-805 (1994)).

While GFP has been used as a fluorescent label in protein localizationand conformation studies (Heim, R., et al., Proc. Natl. Acad. Sci. USA91:1250-1254 (1994); Yokoe, H., and Meyer, T., Nature Biotech.14:1252-1256 (1996)), it has gained increased attention in the field ofmolecular genetics since the demonstration of its utility as a reportergene in transfected prokaryotic and eukaryotic cells (Chalfie, M., etal., Science 263:802-805 (1994); Heim, R., et al., Proc. Natl. Acad.Sci. USA 91:1250-1254 (1994); Wang, S., and Hazelrigg, T., Nature369:400-403 (1994)). GFP has also been used in fluorescence resonanceenergy transfer studies of protein-protein interactions (Heim, R., andTsien, R. Y., Curr. Biol. 6:178-182 (1996)). Since GFP is naturallyfluorescent, exogenous substrates and cofactors are not necessary forinduction of fluorescence, thus providing GFP an advantage over thebiochemical, luminescent and other fluorescent reporter genes describedabove. Visualization of GFP fluorescence does not require the fixationsteps necessary with biochemical reporters such as β-gal and β-glu, nordoes it require extraction from the cell prior to assay as may berequired with luciferase; thus, GFP is suitable for use in proceduresrequiring continued viability of transfected cells. In addition, sincethe GFP cDNA containing the complete coding region is less than 1kilobase in size (Prasher, D. C., et al., Gene 111:229-233 (1992)), itis easily manipulated and inserted into a variety of vectors for use increating stable transfectants (Chalfie, M., et al., Science 263:802-805(1994)).

Despite these advantages, however, the use of wildtype GFP has a fewlimitations. For example, the excitation and emission maxima of wildtypeGFP are not within the range of wavelengths of standard fluorescenceoptics (at which GFP demonstrates relatively low quantum yield (i.e.,low intensity of fluorescence)). In addition, GFP shows low efficiencyof transcription in mammalian cells upon transfection and is packagedinto low-solubility inclusion bodies in bacteria (thus providingdifficulty in purification). These limitations have been overcome to alimited extent via the introduction of selected point mutations into thesequence of wildtype GFP.

GFP Mutants

One of the earliest mutation studies of GFP, in which the tyrosineresidue at position 66 in the wildtype protein (“wt-GFP”) was replacedwith a histidine residue, resulted in a mutant protein which fluorescedblue instead of green when excited with ultraviolet (UV) light (Heim,R., et al., Proc. Natl. Acad. Sci. USA 91:1250-1254 (1994)). This mutantprotein not only provided a capacity for two distinguishable wavelengthsfor use in studies comparing independent proteins and gene expressionevents, but also demonstrated that single point mutations in GFP couldinduce drastic changes in the photochemistry of the protein. Three othersets of specific point mutations have been shown to increase theexcitation and emission maxima of GFP such that they fall well withinthe range of standard fluorescein optics (Ehrig, T., et al., FEBS Letts.367:163-166 (1995); Delagrave, S., et al., Bio/Technology 13:151-154(1995); Heim, R., and Tsien, R., Curr. Biol. 6:178-182 (1996)), thuspermitting the use of GFP with standard laboratory fluorescencedetection systems. The problem of low quantum yield by wt-GFP has beenpartially addressed by mutating the serine residue at position 65 to athreonine (“S65T”), either without (Heim, R., et al., Proc. Natl. Acad.Sci. USA 91:1250-1254 (1994)) or with (Cormack, B., et al., Gene173:33-38 (1996)) a concomitant mutation at position 64, or by mutatingother residues in the non-chromophore region (Crameri, A., et al.,Nature Biotech. 14:315-319 (1996)). The S65T mutation also appears toimprove the rate of fluorophore formation in transfected cells byapproximately four-fold over wt-GFP, thus allowing earlier and moresensitive detection of transfection with this mutant than with wt-GFP(Heim, R., et al., Proc. Natl. Acad. Sci. USA 91:1250-1254 (1994)). Bycombining the S65T mutation with a mutation at position 64 replacingphenylalanine with leucine, approximately 90% of the mutant GFPexpressed in bacteria is soluble, thus improving protein purificationand yields (Cormack, B., et al., Gene 173:33-38 (1996)). Another seriesof mutations results in a mutant fusion GFP consisting of linked blueand green-fluorescing proteins which have proven useful in studies ofprotein localization, targeting and processing (Heim, R., and Tsien, R.Y., Curr. Biol. 6:178-182 (1996)). Analogously, chimeric constructscomprising GFP linked to other proteins have been used in studies of ionchannel expression and function (Marshall, J., et al., Neuron 14:211-215(1995)), and in organelle targeting studies where they have provided ameans for selectively and distinctively labeling the organelles ofliving cells (Rizzuto et al., Curr. Biol. 6:183-188 (1996)). Finally, bycombining the S65T mutation with other mutations throughout thenonchromophore regions of the wt-GFP gene, a “humanized” mutant GFP (SEQID NOs:1, 2) has been produced that not only shows a significantincrease in fluorescence intensity and rate of fluorophore formationover wt-GFP (via the S65T mutation) but also demonstrates a 22-foldincreased expression efficiency in mammalian cells (Evans, K., et al.,FOCUS 18(2):40-43 (1996); Zolotukhin, S., et al., J. Virol. 70:4646-4654(1996)). This humanization was achieved via 92 base substitutions (in 88codons) to the wt-GFP gene which were amino acid-conservative and whichwere made to provide a pattern of codon usage more closely resemblingthat of mammalian cells, as opposed to the jellyfish codon patternsfound in the wt-GFP gene which are less efficiently translated inmammalian cells. A summary of these GFP chromophore mutants is presentedin Table 1. TABLE 1 GFP Chromophore Mutants. Amino Acid Residue Number:Mutant 64 65 66 Reference¹ (Wildtype) Phe Ser Tyr Prasher et al., 1992GreenLantern-1 Phe Thr Tyr Evans et al., 1996 Humanized GFP Phe Thr TyrZolotukhin et al., 1996 Y66H Phe Ser His Heim et al., 1994 Y66W Phe SerTrp Y66F Phe Ser Phe RSGFP1 Gly Ser Tyr Delagrave et al., 1995 RSGFP2Leu Leu Tyr RSGFP3 Gly Cys Tyr RSGFP4 Met Gly Tyr RSGFP6 Val Ala TyrRSGFP7 Leu Cys Tyr S65A Phe Ala Tyr Heim et al., 1996 S65L Phe Leu TyrS65C Phe Cys Tyr S65T Phe Thr Tyr GFPmut1 Leu Thr Tyr Cormack et al.,1996¹See preceding text for full citations.

Despite some success in overcoming certain of the above-describedlimitations of GFPs, the sensitivity of GFP as a reporter gene (measuredas percentage of positive cells) is not as high as that of standardbiochemical reporter genes such as β-gal (Evans, K., et al., FOCUS18(2):40-43 (1996)). In addition, the use of GFP as a reporter gene or aprotein tag requires the use of fluorescent excitation and emissionoptics, which increases user expense and which is more technicallychallenging than the use of visible or white light optics often usedwith standard reporters such as β-gal. Thus, a need currently exists foradditional GFP variants which are more highly fluorescent, humanized,rapidly expressed in mammalian cells, capable of being observed usingstandard white light optics, and which provide an increased level ofsensitivity.

SUMMARY OF THE INVENTION

It is thus an object of the present invention to provide mutant GFPcDNAs and proteins. In one aspect, the invention relates to such mutantGFP cDNAs which, when transfected into prokaryotic (e.g., bacterial) oreukaryotic (e.g., mammalian) cells, increase the sensitivity ofdetection (measured as percentage or number of positive cells). Thepresent invention thus provides nucleic acid molecules encoding mutantGFPs, wherein the mutant GFPs have an amino acid sequence comprising anamino acid residue lacking an aromatic ring structure at position 64 andan amino acid residue having a side chain no longer than two carbonatoms in length at position 65. Preferably, (a) if the residue atposition 64 is leucine then the residue at position 65 is not cysteineor threonine; (b) if the residue at position 64 is valine then theresidue at position 65 is not alanine; (c) if the residue at position 64is methionine then the residue at position 65 is not glycine; and (d) ifthe residue at position 64 is glycine then the residue at position 65 isnot cysteine. The invention is particularly directed to such nucleicacid molecules encoding mutant GFPs wherein the amino acid residue atposition 64 is alanine, valine, leucine, isoleucine, proline,methionine, glycine, serine, threonine, cysteine, alanine, asparagine,glutamine, aspartic acid or glutamic acid, most preferably cysteine ormethionine. The invention is also particularly directed to such nucleicacid molecules encoding mutant GFPs wherein the amino acid residue atposition 65 is alanine, glycine, threonine, cysteine, asparagine oraspartic acid, most preferably alanine. In particular, the inventionprovides nucleic acid molecules encoding mutant GFPs wherein the aminoacid at position 64 is cysteine or methionine and the amino acid atposition 65 is alanine, and nucleic acid molecules encoding mutant GFPshaving an amino acid sequence as set forth in either SEQ ID NO:5 or SEQID NO:6.

In additional aspects, the invention provides mutant GFPs encoded by anyof the above-described nucleic acid molecules, vectors (particularlyexpression vectors) comprising these nucleic acid molecules, host cells(prokaryotic or eukaryotic (including mammalian)) comprising thesenucleic acid molecules or vectors, and compositions comprising plasmidpGreenLantern-2/A1 or plasmid pGreenLantern-2/A4. The invention alsoprovides methods for producing a mutant GFP, comprising culturing theabove-described host cells under conditions favoring the production of amutant GFP and isolating the mutant GFP from the host cell. Theinvention also provides mutant GFPs produced by these methods,particularly wherein the mutant GFPs emit fluorescent light whenilluminated with white light. The invention also relates to compositionscomprising the above-described mutant GFPs.

The invention is further directed to kits for transfecting a host cellwith the nucleic acid molecules encoding the present mutant GFPs, suchkits comprising at least one container containing a nucleic acidmolecule encoding a mutant GFP such as those described above, whichpreferably comprises plasmid pGreenLantern-2/A1 or plasmidpGreenLantern-2/A4. These kits of the invention may optionally furthercomprise at least one additional container containing a reagent,preferably comprising a liposome and most preferably LIPOFECTAMINE™, fordelivering a mutant GFP nucleic acid molecule into a host cell.

The invention is further directed to kits for labeling a polypeptidewith the present mutant GFPs, such kits comprising at least onecontainer containing a mutant GFP such as those described above,preferably a mutant GFP having an amino acid sequence as set forth inSEQ ID NO:5 or SEQ ID NO:6. These kits of the invention may optionallyfurther comprise at least one additional container containing a reagentfor covalently linking this mutant GFP to the target polypeptide.

The fluorescence of all of the GFP mutants provided by the presentinvention is observable with fluorescein optics, making these mutantproteins amenable to use in techniques such as fluorescence microscopyand flow cytometry using standard FITC filter sets. In addition, thefluorescence of certain of the present GFP mutants, particularly thosehaving amino acid sequences as set forth in SEQ ID NOs: 5 and 6, isvisible using standard white light optics (e.g., incandescent orfluorescent indoor lighting, or sunlight). The nucleic acid moleculesand mutant GFPs provided by the present invention thus contributeimproved tools for detection of transfection, for fluorescent labelingof proteins, for construction of fusion proteins allowing examination ofintracellular protein expression, biochemistry, and trafficking, and forother applications requiring the use of reporter genes.

Other preferred embodiments of the present invention will be apparent toone of ordinary skill in light of the following drawings and descriptionof the invention, and of the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a depiction of the nucleotide (SEQ ID NO: 1) and deduced aminoacid (SEQ ID NO:2) sequences of humanized S65T mutant A. victoria GreenFluorescent Protein cDNA (after Zolotukhin, S., et al., J. Virol.70:4646-4654 (1996)).

FIG. 2 is a depiction of the nucleotide (SEQ ID NO:3) and deduced aminoacid (SEQ ID NO:4) sequences of A. victoria Green Fluorescent ProteincDNA (after Prasher, D. C., et al., Gene 111:229-233 (1992)).

FIG. 3 is a depiction of the amino acid sequence (SEQ ID NO:5) of the A1GFP mutant.

FIG. 4 is a depiction of the amino acid sequence (SEQ ID NO:6) of the A4GFP mutant.

FIG. 5 is a structural map of plasmid pGreenLantern-1.

FIG. 6 is a structural map of plasmid pGreenLantern-2.

FIG. 7 is a fluorescence photomicrograph of CHO-K1 cells viewed 24 hoursafter transfection with the A1 GFP mutant (plasmid pGreenLantern-2/A1).

FIG. 8 is a fluorescence photomicrograph of CHO-K1 cells viewed 24 hoursafter transfection with the A4 GFP mutant (plasmid pGreenLantern-2/A4).

FIG. 9 is a fluorescence photomicrograph of negative control CHO-K1cells viewed 24 hours after transfection with the pGreenLantern-2backbone.

FIG. 10 is a bar graph demonstrating the fluorescence of CHO-K1 cellsdetermined by flow cytometry 24 hours after transfection with variousGFP mutants.

FIG. 11 is a bar graph demonstrating the fluorescence of CHO-K1 cellsdetermined by flow cytometry 48 hours after transfection with variousGFP mutants.

FIG. 12 is a structural map of plasmid pProEX HTb.

DETAILED DESCRIPTION OF THE INVENTION

Overview

The present invention provides nucleic acid molecules encoding mutantGFPs, vectors and host cells comprising these nucleic acid molecules,the mutant GFP polypeptides, and methods for producing mutant GFPs.Although specific plasmids, vectors, promoters, selection methods andhost cells are disclosed and used herein and in the Examples, otherpromoters, vectors, selection methods and host cells, both prokaryoticand eukaryotic, are well-known to one of ordinary skill in the art andmay be used to practice the present invention without departing from thescope of the invention or any of the embodiments thereof.

In the present invention, GFPs with selective point mutations at aminoacid positions 64 and 65 have been constructed and analyzed. In general,it has been discovered in the present invention that when the amino acidresidue at position 64 (phenylalanine in wt-GFP) is mutated to an aminoacid lacking an aromatic ring (e.g., alanine, valine, leucine,isoleucine, proline, methionine, glycine, serine, threonine, cysteine,asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine orhistidine), an increase in fluorescence quantum yield is observed.Increased fluorescence intensity is also observed when the amino acidresidue at position 65 (serine in wt-GFP) is mutated to an amino acidhaving a side chain consisting of no more than two carbon atoms (e.g.,alanine, glycine, threonine, cysteine, asparagine or aspartic acid),which induce a significant “red-shift” in excitation maximum fromultraviolet to visible blue wavelengths and a single excitation maximuminstead of a dual excitation maximum as in the wildtype protein.Together, these general results indicate that in order to construct GFPmutants with a dramatic increase in fluorescence intensity from wt-GFP,either position 64 or position 65 should contain a reactive amino acid,although particular amino acids appear to be preferred at each positionas described below. Furthermore, it has been unexpectedly discoveredthat several of the mutant GFPs of the present invention, unlike thosepreviously known in the art, will emit fluorescence when illuminated bywhite light (e.g., incandescent or fluorescent indoor lighting, orsunlight).

Accordingly, in the present invention, specific mutations are introducedinto positions 64 and 65 of the wt-GFP cDNA sequence (SEQ ID NO:3).Alternatively, increased expression of the present mutant GFPs may beobtained by introducing the preferred mutations into a humanized GFPgene such as that described previously (SEQ ID NO: 1) (Evans, K., etal., FOCUS 18(2):40-43 (1996); Zolotukhin, S., et al., J. Virol.70:4646-4654 (1996)).

Construction of GFP Mutants

Preparation of GFP Plasmids

The wt-GFP may be cloned from its natural source, Aequorea victoria, asdescribed (Prasher, D. C., et al., Gene 111:229-233 (1992)). Morepreferably, GFP cDNA to be mutated is contained within a plasmidconstruct or vector, preferably an expression vector, suitable for usein transfecting mammalian cells, such as pRAY-1 wherein the wt-GFP cDNAis under the control of the human cytomegalovirus (CMV)enhancer/promoter (Marshall, J., et al., Neuron 14:211-215 (1995)). Mostpreferably, to provide for optimum expression of the mutant GFPs inmammalian cells, the humanized S65T mutant GFP cDNA (Evans, K., et al.,FOCUS 18(2):40-43 (1996); Zolotukhin, S., et al., J. Virol. 70:4646-4654(1996)) under control of the CMV enhancer/promoter may be used,contained in plasmid pGreenLantern-1 (FIG. 5), which is availablecommercially from Invitrogen Corporation, Carlsbad, Calif.

The above-described plasmids may be used directly for preparation ofmutant GFP cDNAs according to the present invention. Alternatively, astop codon in the 5′ multiple cloning site of pGreenLantern-1 may beshifted out of frame by oligonucleotide ligation methods to allow themutant GFPs of the present invention to be used in the construction offusions between GFP and other proteins, as described below.

Mutations to GFP cDNA

A variety of random or site-directed mutagenic techniques may be used toprepare the mutant GFPs of the present invention. Appropriate methodsinclude chemical mutagenesis using, for example, sodium bisulfite orhydroxylamine (Myers, R. M., et al., Science 229:242-247 (1985);Sikorski, R. S., and Boeke, J. D., Meth. Enzymol. 194:302-318 (1991)),linker insertion mutagenesis (Heffron, F., et al., Proc. Natl. Acad.Sci. USA 75:6012-6016 (1978)), deletion mutagenesis (Lai, C. J., andNathans, D., J. Mol. Biol. 89:179-193 (1974); McKnight, S. L., andKingsbury, R., Science 217:316-324 (1982)), enzyme misincorporationmutagenesis (Shortle, D., et al., Proc. Natl. Acad. Sci. USA79:1588-1592 (1982)), oligonucleotide-directed mutagenesis (Hutchinson,C. A., et al., J. Biol. Chem. 253:6551-6560 (1978); Zoller, M. J., andSmith, M., Nucl. Acids Res. 10:6487-6500 (1982); Taylor, J. W., et al.,Nucl. Acids Res. 13:8765-8785 (1985)), and cassette mutagenesis (Lo,K.-M., et al., Proc. Natl. Acad. Sci. USA 81:2285-2289 (1984); Wells, J.A., et al., Gene 34:315-323 (1985)). To improve the fidelity andefficiency of mutagenesis, the use of the polymerase chain reaction(PCR) in accomplishing GFP mutagenesis by one or more of the foregoingmethods is preferred (Higuchi, R., et al., Nucl. Acids Res. 16:7351-7367(1988); Leung, D. W., et al., Technique 1:11-15 (1989); Clackson, T.,and Winter, G., Nucl. Acids Res. 17:10163-10170 (1989)).

Most preferably, mutations are made to GFP cDNA by uracil DNAglycosylase (UDG) mutagenesis using PCR amplification (Nisson, P., etal., PCR Meth. Appl. 1:120-123 (1991)). In this approach, the plasmidcontaining GFP cDNA, most preferably pGreenLantern-1 comprisinghumanized S65T GFP (FIG. 5), is used as the PCR template, and a sense orantisense primer consisting essentially of an oligonucleotide containingat least one mismatched nucleotide (available commercially fromInvitrogen Corporation, Carlsbad, Calif.) is added to the reactionmixture. Amplification reaction mixtures most preferably contain 1×PCRbuffer, about 10 micromolar each of deoxyATP, deoxyTTP, deoxyCTP anddeoxyGTP, about 25 picomoles each of sense and antisense primers andabout 10 nanograms of template. PCR is performed by techniques that areroutine in the art, and after at least five PCR cycles, samples of thereaction mixture are treated with UDG, most preferably for 30 minutes at37° C., as described (Nisson, P., et al., PCR Meth. Appl. 1:120-123(1991)).

The mutated GFP nucleic acid molecules preferably will comprise nucleicacid sequences encoding mutant proteins in which one or more amino acidresidues have been mutated from the wildtype amino acid sequence setforth in FIG. 2 and SEQ ID NO:4. Such mutations may include, forexample, substitutions, deletions, insertions or modifications, andpreferably are amino acid substitutions. Particularly preferred areamino acid substitutions occurring in the three amino acid chromophoreof GFP at residues 64, 65 and 66 of the wildtype GFP sequence (FIG. 2and SEQ ID NO:4), wherein the phenylalanine residue at position 64(Phe64), the serine residue at position 65 (Ser65), and the tyrosineresidue at position 66 (Tyr66), are each individually, or all together,replaced by other amino acid residues. More preferred mutant GFPs of theinvention include, but are not limited to, those with the followingsubstitutions from the wildtype GFP sequence shown in FIG. 2 and SEQ IDNO:4:

-   -   serine 65 replaced by threonine (Ser65→Thr);    -   Phe64→Cys and Ser65→Ala (SEQ ID NO:5);    -   Phe64→Cys and Ser65→Thr;    -   Phe64→Leu and Ser65→Thr;    -   Phe64→Met and Ser65→Ala (SEQ ID NO:6);    -   Phe64→Met and Ser65→Thr;    -   Phe64→Met, Ser65→Phe and Tyr66→Phe;    -   Phe64→Met, Ser65→Phe and Tyr66→Lys;    -   Phe64→Thr and Ser65→Cys; and    -   Phe64→Val and Ser65→Cys

Other suitable mutations and mutant GFP amino acid sequences may bedetermined by one of ordinary skill without undue experimentationaccording to the methods described herein and others that are known inthe art. As a practical matter, whether a particular mutation orcombination of mutations produces a mutant GFP that may have theabove-described desirable properties (e.g., higher expression inmammalian cells, higher fluorescence intensity under UV or white lightillumination) may be determined by one of ordinary skill using themutation, transfection, expression and detection methods described indetail below in the Examples, as well as using standard techniques thatare routine in the art.

Following mutagenesis by any of the above-described methods, theresulting nucleic acid molecules encoding the mutant GFPs may beinserted into one or more vectors, such as those described above, whichare preferably expression vectors. A particularly preferred vector forcontaining the present mutant GFP nucleic acid molecules isp-GreenLantern-2 (FIG. 6). Methods for producing the mutant GFP-vectorconstructs will be familiar to those of ordinary skill, and are providedin detail below in Example 1.

Once they have been constructed, the vectors comprising the mutant GFPnucleic acid molecules may be formulated into a variety of compositions,such as solutions (e.g., buffer solutions) to be used in transfectinghost cells. Alternatively, the vector constructs may be purified andstored according to standard techniques for handling recombinant DNAplasmid vectors (Sambrook, J., et al., Molecular Cloning, a LaboratoryManual, 2nd Ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress, pp. 1.3-1.20 (1989)).

More preferably, the mutant GFP-containing plasmid vectors aretransformed into a competent host cell. Any competent host cell may beused, including those of bacteria (e.g., E. coli), yeast (e.g.,Saccharomyces spp.), insects (e.g., Spodoptera spp.) and mammals (e.g.,CHO or BHK cells), although a competent strain of E. coli such as DH10B(Invitrogen Corporation, Carlsbad, Calif.) is most preferably used.Transformation of mutagenized GFP cDNAs into host cells may beaccomplished by any technique generally used for introduction ofexogenous DNA, including the chemical, viral, electroporation,lipofection and microinjection methods that are well-known in the art.Particularly preferred methods for transformation includeelectroporation and liposome-mediated transfection (lipofection), thelatter most preferably being accomplished using LIPOFECTAMINE™(Invitrogen Corporation, Carlsbad, Calif.).

After expansion of transformed cultures, mutated GFP cDNA is isolatedfrom the host cells by routine methods (Sambrook, J., et al., MolecularCloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press, pp. 1.21-1.52 (1989)) and is subclonedinto a plasmid backbone for use in subsequent transfections. Mostpreferably, this plasmid backbone is the pGreenLantern-2 backbone (seeFIG. 6) which contains a universal sequencing primer downstream from aCMV enhancer promoter and an NsiI site immediately upstream of the CMVpromoter allowing excision of the promoter region, along with XbaI, XhoIand HindIII sites in place of the 3′ NotI site in pGreenLantern-1 (FIG.4).

Fusion sequences of GFP cDNA with nucleotide sequences encoding proteinsof interest may be prepared by cloning the desired sequence(s) intopGreenLantern-2 at the 5′ multiple cloning site using standardtechniques. These fusion constructs allow the use of the mutant GFPs ofthe present invention as reporters of transfection efficiency. Inaddition, fusion constructs such as these will allow a directexamination of the expression, biochemistry and localization of thefused proteins intracellularly.

Alternatively, to examine the structure and function of regulatorysequences (e.g., promoters, enhancers, inhibitors) in native genes, theGFP mutant cDNAs may be directly transfected or inserted, using routinemethods, into target, genomic or extrachromosomal DNA sequences in hostcells (Chalfie, M., et al., Science 263:802-805 (1994)).

Transfection of Hosts With GFP Mutants

Target cells to be transfected with cDNAs comprising mutant GFPs (eitherfused or unfused to accessory sequences) are grown and maintained inculture according to routine methods. Cells may be transfected withmutant GFP cDNA by any method described above, although electroporationor liposome-mediated transfection (particularly using LIPOFECTAMINE™)are preferred. Following transfection, cells are incubated for 12-48hours, preferably 18-24 hours and most preferably for about 24 hours.Transfected cells may then be examined for the expression of mutant GFP,manifested as green intracellular fluorescence. With standard opticalfilters routinely used for examining fluorescein (typically excitationwavelength of about 475 nm, dichroic filter of 485 nm, emissionwavelength of about 490 nm), this fluorescence may be examinedqualitatively, for example by fluorescence microscopy, orquantitatively, for example by spectrofluorimetry or flowcytofluorimetry. In addition, transfected cells expressing relativelyhigh amounts of mutant GFPs of the present invention may be separatedfrom non-transfected cells, or from those expressing lower levels ofGFP, by fluorescence-based single cell separation techniques such asfluorescence-activated cell sorting. Alternatively, transfected cellsexpressing mutant GFPs that fluoresce under white light illumination,particularly those having amino acid sequences as set forth in SEQ IDNOs: 5 and 6, may be examined by the above-described qualitative andquantitative methods using standard white light optics (e.g.,incandescent or halogen lighting, or sunlight).

These transfected host cells may also be used in methods for theproduction of mutant GFPs of the invention. Such methods may comprise,for example, culturing the above-described host cells under conditionsfavoring the production of the mutant GFPs by the host cells, andisolating the mutant GFPs from the host cells and/or the culture mediumin which the host cells are cultured. Typical host cell cultureconditions favoring production of recombinant proteins, such as thepresent mutant GFPs, are well-known in the art (see, e.g., Sambrook, J.,et al., Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press (1989)). The mutantGFPs produced by these methods may then be isolated by any of a numberof protein purification techniques, such as chromatography (preferablyaffinity chromatography, HPLC or FPLC), salt extraction (such asammonium sulfate precipitation), electrophoresis, dialysis, or acombination thereof, to produce isolated mutant GFPs of the invention.These mutant GFPs may then be stored until use (preferably attemperatures below 0° C., more preferably at about −20° C. to about −70°C.), or they may be formulated into compositions. Preferred suchcompositions may comprise, for example, one or more of the mutant GFPsof the invention and one or more additional components, such as one ormore buffer salts, one or more inorganic salts or ions thereof, one ormore detergents, one or more preservatives, and the like, preferably inan aqueous or organic solvent.

Detection Methods

In additional embodiments, the invention relates to methods of detectingthe presence of a mutant GFP, or of a cell (such as a prokaryotic oreukaryotic, including mammalian, cell) expressing a mutant GFP. Suchmethods of the invention may comprise, for example, illuminating themutant GFP or cell expressing the mutant GFP with a source of whitelight under conditions such that the mutant GFP or cell expressing themutant GFP emits visible fluorescent light. In the present methods, theillumination source may be any light source emitting white (i.e.,visible) light, including but not limited to an incandescent lightsource, a fluorescent light source, a halogen light source, sunlight,and the like. When illuminated by such a white light source, mutantGFPs, such as those of the present invention, will emit fluorescentlight of various visible wavelengths (depending upon the specificmutations contained in the mutant GFP, as described above), which may bedetected by eye or by any of the above-described qualitative orquantitative mechanical means.

Kits

In other preferred embodiments, the compositions of the presentinvention may be assembled into kits for use in transfecting host cellswith the nucleic acid molecules encoding the present mutant GFPs, or forlabeling target polypeptides with the present mutant GFPs. Host celltransfection kits according to the present invention may comprise atleast one container containing one or more of the above-describednucleic acid molecules encoding a mutant GFP (or a compositioncomprising one or more of the nucleic acid molecules or plasmidsdescribed above), which nucleic acid molecule preferably comprisesplasmid pGreenLantern-2/A1 or plasmid pGreenLantern-2/A4 (see Example 1below). These transfection kits of the invention may optionally furthercomprise at least one additional container which may contain, forexample, a reagent for delivering the mutant GFP nucleic acid moleculeinto a host cell; in preferred kits, this reagent may comprise aliposome and most preferably LIPOFECTAMINE™. Polypeptide labeling kitsaccording to the present invention may comprise at least one containercontaining, for example, a mutant GFP such as those described above (ora composition of the invention comprising a mutant GFP), which ispreferably a mutant GFP having an amino acid sequence as set forth inSEQ ID NO:5 or SEQ ID NO:6. These labeling kits of the invention mayoptionally further comprise at least one additional container which maycontain, for example, a reagent for covalently linking the mutant GFP tothe target polypeptide.

Use of Mutant GFPs

The mutant GFPs and kits of the present invention may be used in avariety of applications. For example, the mutant GFP cDNAs are useful asreporter genes that allow a determination of transfection efficiency andsuccess (Chalfie, M., et al., Science 263:802-805 (1994)).Alternatively, the mutant proteins themselves may be used as fluorescentlabels suitable for detectably labeling other proteins, nucleic acids orparticulates to be used in a variety of applications (Heim, R., et al.,Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994); Yokoe, H., and Meyer,T., Nature Biotech. 14:1252-1256 (1996)), such as labeling antibodiesused in infectious disease diagnostic methods; mutant GFPs may beattached to target polypeptides and proteins by a variety of methodsthat are well-known to one of ordinary skill in the art, including theuse of chemical coupling reagents. In addition, fusion complexes betweenGFP and other proteins may be constructed to allow closer and moresensitive determinations of the expression, biochemistry, localizationand trafficking of intracellular proteins in many host cells (Heim, R.,et al., Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994); Wang, S., andTulle, H., Nature 369:400-403 (1994); Marshall, J., et al., Neuron14:211-215 (1995); Rizzuto, R., et al., Curr. Biol. 6:183-188 (1996)).Importantly, use of the mutant GFPs that emit fluorescence whenilluminated by white light will spare the user considerable expense andtechnical difficulty that can accompany the use of fluorescent opticsfor the examination of fluorescent reporter genes such as GFP.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

EXAMPLES Example 1 Construction of Mutant GFP cDNAs

Plasmids. As depicted in FIG. 5, pGreenLantern-1 (InvitrogenCorporation, Carlsbad, Calif.; catalogue no. 10642) contains thehumanized S65T mutant GFP cDNA (FIG. 1; SEQ ID NOs:1, 2) (Evans, K., etal., FOCUS 18(2):40-43 (1996); Zolotukhin, S., et al., J. Virol.70:4646-4654 (1996)). This plasmid serves as the source of the GFP DNAsequence used for mutagenesis. As depicted in FIG. 6, pGreenLantern-2contains a universal sequencing primer downstream of the CMV promoteralong with an NsiI site immediately upstream of the CMV promoterallowing excision of the promoter region. It also contains XbaI, XhoIand HindIII sites in place of the 3′ NotI site in pGreenLantern-1. Astop codon in the 5′ multiple cloning site of pGreenLantern-1 wasshifted out of frame to allow possible fusions to GFP inpGreenLantern-2.

Mutations to GFP cDNA by UDG cloning. PCR was performed in an MJResearch DNA Engine™ thermal cycler using the following conditions: 94°C. for 60 seconds, 94° C. for 30 seconds, 55° C. for 30 seconds and 72°C. for 4 minutes, repeated for 20 cycles. Sense oligonucleotide primerscontaining specific mismatches to the wt-GFP sequence (SEQ ID NOs:7-15;Table 2) were obtained from Invitrogen Corporation (Carlsbad, Calif.).TABLE 2 Sense Oligonucleotides Used for UDG Cloning Mutations. AminoAcid Single-stranded Muta- Oligonucleotide SEQ ID Vector tions Sequence(5′ to 3′) NO: pGreenLantern-2/A1 Cys64, CAACACUGGUCACUACCTG- 7 Ala65CGCCTATGGCGTGC pGreenLantern-2/A2 Cys64, CCAACACUGGUCACUACCT- 8 Thr65GCACCTATGG pGreenLantern-2/A3 Leu64, CAACACUGGUCACUACCCT- 9 Thr65CACCTATGGCGTGCAGT pGreenLantern-2/A4 Met64, CAACACUGGUCACUACAAT- 10Ala65 GGCCTATGGCGTGCAGTGCT pGreenLantern-2/A5 Met64,CAACACUGGUCACUACCAT- 11 Thr65 GACCTATGGCGTGCAGTGCT pGreenLantern-2/A6Met64, CAACACUGGUCACUACCAT- 12 Phe65, GTTCTTCGGCGTGCAGTGCT Phe66pGreenLantern-2/A7 Met64, CAACACUGGUCACUACCAT- 13 Phe65,GTTCAAGGGCGTGCAGTGCT Lys66 pGreenLantern-2/A8 Thr64,CAACACUGGUCACUACCAC- 14 Cys65 ATGCTATGGCGTGCAGT pGreenLantern-2/A9Val64, CAACACUGGUCACUACCGT- 15 Cys65 GTGCTATGGCGTGCAGT

The antisense oligonucleotide primer used for each mutation set had thefollowing sequence: 5′-AGU-GAC-CAG-UGU-UGG-CCA-AGG-CAC-AGG-GAG-CTT-3′(SEQ ID NO:16). The template plasmid used was pGreenLantern-1 (FIG. 5)with a universal reverse sequencing primer incorporated into thebackbone. Amplifications reactions contained 1×PCR buffer, 10 micromolardeoxynucleoside triphosphates, 25 picomoles of each primer (sense andantisense) and 10 nanograms of template DNA in a 50 microliter volume.After 6, 9 and 20 PCR cycles were completed, 10 microliter samples weretaken and checked via agarose gel electrophoresis for excess background.Two. 20 microliter samples of each 6-cycle aliquot were digested withDpnI at 37° C. for 30 minutes, then at 75° C. for 15 minutes and allowedto cool to room temperature. One of the samples from each reaction (foursamples in all) was treated with one unit of uracil DNA glycosylase(UDG) at 37° C. for 30 minutes (Nisson, P., et al., PCR Meth. Appl.1:120-123 (1991)). PCR samples were then transformed into 100microliters of MAX Efficiency DH10B™ Competent Cells (InvitrogenCorporation; Carlsbad, Calif.). The mutated portion of the GFP cDNA wasthen subcloned with a NotI and BamHI digest into the pGreenLantern-2backbone (FIG. 6) which was not subjected to PCR (Sambrook, J., et al.,Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (1989)). This approach yieldednine separate mutant GFP plasmid vectors, designated pGreenLantern-2/A1through pGreenLantern-2/A9 (Table 2), each with a specific mutation orset of mutations within the GFP chromophore region at amino acids 64-66.

Example 2 Growth and Transfection of Host Cells With Mutant GFPs

Cell Culture. Chinese hamster ovary cells (CHO-K1, obtained fromAmerican Type Culture Collection (ATCC), Rockville, Md.) were culturedin D-MEM (4,500 milligrams/liter D-glucose with L-glutamine and phenolred) plus 10% fetal bovine serum (FBS), 0.1 millimolar nonessentialamino acids, 2.5 units per milliliter penicillin and 2.5 micrograms permilliliter streptomycin (Freshney, R. I., Culture of Animal Cells: AManual of Basic Techniques, 3rd Ed., New York: Wiley-Liss (1994)). Cellswere grown at 37° C. in a 5% CO₂/air incubator. All media and reagentswere from Invitrogen Corporation, Carlsbad, Calif.

Transfection. CHO-K1 cells were plated at 2×10⁵ cells per well intosix-well (35 millimeter diameter) plates one day prior to transfection.Immediately before transfection, cells were rinsed with mediumcontaining no serum or antibiotics. LIPOFECTAMINE™ reagent was dilutedinto 100 microliters of OPTI-MEM-I Reduced Serum Medium (without FBS) togive a final concentration of LIPOFECTAMINE of 6 microliters per well.DNA was diluted separately to a concentration of 1 microgram per well in100 microliters of OPTI-MEM-I. Transfection complexes were formed bycombining diluted lipid and DNA and incubating for 30 minutes prior toaddition to cells. Transfection complexes were then diluted 1:5 withD-MEM containing no FBS or antibiotics and added to the rinsed cells.Cells were transfected for five hours at 37° C., then fed with an equalvolume of D-MEM containing 20% FBS, 0.1 millimolar nonessential aminoacids, and no antibiotics. Cells were grown overnight at 37° C., 5%CO₂/air. In some studies, cells were grown for 48 hours; in thesestudies, transfection complexes were removed from cells 24 hours afteraddition and cells were fed with 2 milliliters per well of completemedium.

Regardless of the vector used, host cells transfected with the mutantGFP genes demonstrated approximately equivalent growth rates as controlcells transfected with the wildtype GFP gene or with other reportergenes (e.g., β-gal). These results indicate that transfection with themutant GFP cDNAs of the present invention does not adversely affect thegrowth or culturability of the host cells more than transfection withany other reporter vector.

Example 3 Characterization of GFP Mutants Expressed in Eukaryotic Cells

Formalin Fixation. Transfected host cells were rinsed in Dulbecco'sPhosphate Buffered Saline (PBS), then fixed in a solution of 10%formalin in PBS for one hour. Formalin was then removed, and cells wererinsed and stored in PBS at 4° C. until being analyzed.

Fluorescence Microscopy. Formalin-fixed cells were examined andphotographed using an inverted phase contrast fluorescence microscopeequipped with FITC filters (excitation 475 nm/dichroic 485 nm/barrier490 nm) and a 50 watt mercury arc bulb at 1.25 volts. A 40×-poweradjustable non-phase objective was used for all micrographs, which weretaken through blue, neutral and FITC filters using Kodak Ektachrome ASA400 Daylight (for slides) or Kodak Gold ASA 400 Daylight (for prints).All exposures were for 12 seconds to allow unbiased comparison offluorescence intensity.

Flow Cytofluorimetry. Flow cytofluorimetry was performed on transfectedCHO-K1 cells that were trypsinized and suspended in PBS plus 10%formalin at a concentration of less than 10⁶ cells per milliliter.Measurements were made on a Coulter EPICS® XL-MCL flow cytometer using a15 megawatt argon ion laser. Filters used were 488 nm excitation, 500 nmdichroic LP/525 nm band pass for FL1 (green channel) and 575 bandpass/600 nm dichroic LP for FL2 (orange channel). Samples consisted of20,000 events using PMT voltages of 100 volts for side scatter andforward scatter, 496 volts for FL1 and 505 volts for FL2, all withintegral gain set to 1.0. Color compensation included 7.9% orange signalin FL1 and 3.2% green signal in FL2.

Results. As shown in Table 3, the GFP mutants of the present inventiondisplayed varying intensities and kinetics of formation in transfectedcells. Two of these mutants, designated “A1” (phenylalanine mutated tocysteine at position 64; serine mutated to alanine at position 65; FIG.3; SEQ ID NO:5) and “A4” (phenylalanine mutated to methionine atposition 64; serine mutated to alanine at position 65; FIG. 4; SEQ IDNO:6) were exceptionally bright. As shown in FIGS. 7-9, CHO cellstransfected with plasmid pGreenLantern-2/A1 (FIG. 7) or with plasmidpGreenLantern-2/A4 (FIG. 8) demonstrated a dramatic increase in greenfluorescence intensity over cells transfected with the humanized S65Tmutation of pGreenLantern-1 (FIG. 9) when viewed at 24 hourspost-transfection using FITC optics. TABLE 3 Effects of Point Mutationson GFP Fluorescence Intensity. Vector Amino Acids Fluorescence ResultsWildtype GFP Phe64, Ser65 λ_(ex) = 395 nm (major), 470 nm (minor); 48hours required for detection S65T Phe64, Thr65 6-fold increase inintensity over wildtype pGreenLantern-1 Phe64, Thr65 22-fold increase inintensity (humanized) over wildtype pGreenLantern-2/A1 Cys64, Ala656-fold increase in intensity over S65T pGreenLantern-2/A2 Cys64, Thr6522-fold increase in intensity over wildtype pGreenLantern-2/A3 Leu64,Thr65 6-fold increase in intensity over S65T pGreenLantern-2/A4 Met64,Ala65 6-fold increase in intensity over S65T pGreenLantern-2/A5 Met64,Thr65 Slight increase in intensity over pGreenLantern-1pGreenLantern-2/A6 Met64, Equivalent to wildtype Phe65, Phe66pGreenLantern-2/A7 Met64, Equivalent to wildtype Phe65, Lys66pGreenLantern-2/A8 Thr64, Cys65 Equivalent to wildtypepGreenLantern-2/A9 Val64, Cys65 Slight increase in intensity overpGreenLantern-1

Other mutants produced in the present studies were less satisfactory(Table 3). For example, mutants A5 (phenylalanine mutated to methionineat position 64; serine mutated to threonine at position 65) and A9(phenylalanine mutated to valine at position 64; serine mutated tocysteine at position 65) gave only slightly better fluorescence than thehumanized S65T mutation of pGreenLantern-1. It is possible that thehighly reactive cysteine at position 65 in mutant A9 may interfere withthe formation of the three amino acid heterocyclic ring required for GFPfluorescence (Cody, C. W., Biochemistry 32:1212-1218 (1993)).

Mutant A2 (phenylalanine mutated to cysteine at position 64; serinemutated to threonine at position 65) was equal in fluorescence to thehumanized S65T pGreenLantern-1 (Evans, K., et al., FOCUS 18(2):40-43(1996); Zolotukhin, S., et al., J. Virol. 70:4646-4654 (1996)), whilemutants A6 (phenylalanine mutated to methionine at position 64; serinemutated to phenylalanine at position 65; tyrosine mutated tophenylalanine at position 66), A7 (phenylalanine mutated to methionineat position 64; serine mutated to phenylalanine at position 65; tyrosinemutated to lysine at position 66) and A8 (phenylalanine mutated tothreonine at position 64; serine mutated to cysteine at position 65)demonstrated a decreased fluorescence intensity and were, in fact,equivalent to wt-GFP. No shift in excitation or emission spectra wasdetected with these three mutants, however, as no fluorescence wasobserved using ultraviolet or rhodamine filter combinations.

These results were also observed via flow cytometry. As shown in FIG.10, CHO-K1 cells transfected with the A1 and A4 mutant GFPs demonstrateda dramatic increase in fluorescence over wildtype and A6-A8 mutantswithin 24 hours of transfection. This high level of fluorescence wasmaintained, particularly for cells transfected with the A4 mutant GFP,for at least 48 hours after transfection (FIG. 11).

Mutations at certain amino acid positions outside the chromophore werealso examined for their effects on GFP fluorescence. Mutation ofGln69→Asn in the A4 mutant resulted in a dramatic decrease influorescence relative to the A4 mutant itself, as did mutation ofVal163→Ala and Ile167→Thr in the A4 mutant.

Together, these results indicate that the most preferable mutations forproviding highly fluorescent, rapidly expressed GFPs are those in whichonly one reactive amino acid is present at either position 64 or 65, asin the A1 (Phe64→Cys; Ser65→Ala; SEQ ID NO:5) and A4 (Phe64→Met;Ser65→Ala; SEQ ID NO:6) mutants.

Example 4 Characterization of GFP Mutants Expressed in Prokaryotic Cells

To examine the efficacy of expressing mutant GFPs in prokaryotic cells,mutant GFP cDNAs were subcloned into the bacterial pProEX HTb vector(FIG. 12). GFP cDNA was excised by NotI and XbaI digestion frompGreenLantern-2 (FIG. 6) containing the mutations at positions 64, 65and/or 66 (mutants A1 through A9) shown in Table 3. The bacterial vectorpProEX HTb (FIG. 12) was also digested with the same enzymes. The pProEXHTb backbone and GFP fragments were ligated, to form the correspondingtransfection vectors containing the respective mutant GFP fragments:pProEXA1, pProEXA2, pProEXA3, pProEXA4, pProEXA5, pProEXA6, pProEXA7,pProEXA8 and pProEXA9. These vectors were then individually transformedinto 100 μl of DH10B E. coli host cells; control cells were alsoprepared that had been transfected with a construct containing the S65Tmutant described in Examples 1-3 above. Cells were plated ontoampicillin/IPTG plates and incubated overnight at 37° C., and colonieswere then picked and screened for fluorescence under long ultraviolet(UV) or blue illumination.

Colonies containing the A1, A2, A3, A4, A5, A9 and S65T mutant GFPs alldemonstrated green fluorescence when illuminated with long UV or bluelight, while those containing the A6, A7 and A8 mutant GFPs demonstratedno fluorescence under these conditions. These results are consistentwith those observed in eukaryotic cells, as shown in Example 3 above,and indicate that mutant GFPs may be successfully transfected into andexpressed in prokaryotic cells.

Example 5 Visible Light Excitation of GFP Mutants

To examine the ability of mutant GFPs to emit fluorescence whenilluminated by white light, E. coli cells were transfected and plated asdescribed above in Example 4. Colonies were then picked and examined forfluorescence upon illumination by incandescent light, fluorescent indoorlighting, or sunlight.

Upon induction of the host cells with EPTG, cells transformed with thevector comprising the A4 GFP mutation unexpectedly exhibited brightgreen light emission under normal daylight conditions, without the needfor excitation with UV light. Similar results were observed for cellstransformed with the A3 mutant GFP. Cells containing the A1 and A5mutant GFPs were also seen to be less (but still observably) fluorescentunder white light illumination. Conversely, only very weak emission oflight was observed under white light illumination in the cellstransformed with the vectors comprising only the S65T, A2 and A9mutations. Cells comprising the A6, A7 and A8 mutations exhibited nofluorescence when illuminated by white light.

When plates containing these mutants were stored in the dark at 4° C.for 38 days, however, all of the colonies except those containing theA6, A7 or A8 mutant GFPs were seen to be more intensely fluorescentunder white light illumination. Colonies containing the A3, A4 and A5mutants were more fluorescent under these conditions than were thosecontaining the A1, A2, A9 and S65T mutants, although all coloniesfluoresced more brightly than they did in freshly plated cells (i.e.,when observed within 24-48 hours of transfection). When these plateswere allowed to warm to room temperature, the fluorescence in coloniescontaining the A1, A2, A9 and S65T mutants decreased, while that incolonies containing the A3, A4 and A5 mutants remained brightlyfluorescent.

It is possible that the increased fluorescence observed in stored platesmay have been due to accumulation of mutant protein in the cells overtime in storage, indicating a dependence of white light fluorescenceupon intracellular concentration of the GFP. To test this notion, a6His-tagged A4 GFP construct prepared and isolated by metal affinitychromatography according to standard techniques (see Ausubel, F. M., etal., in Current Protocols in Molecular Biology, New York: John Wiley &Sons, Inc., pp. 10.11.10-10.11.24 (1996)), was examined for fluorescenceunder blue, red and white light at various protein concentrations insolution. At a concentration of about 1.5 μg/ml, the purified A4 GFP wasbrightly fluorescent under sunlight and fluorescent indoor whitelighting, as well as under blue light; no fluorescence was observed,however, under red light. This highly concentrated A4 GFP solutionbecame nonfluorescent upon boiling, but was at least slightlyfluorescent up to a temperature of about 82° C. When diluted to 0.1μg/ml, however, the A4 GFP solution fluoresced brightly under blue light(closer in wavelength to the excitation maximum of GFP which is in theUV range), but did not fluoresce under white light illumination. Theseresults suggest that the increased fluorescence observed upon whitelight illumination of colonies stored for extended periods of time maybe due to accumulation of GFP protein in the cells.

Taken together, these results indicate that prokaryotic cells containingthe A3 or A4 mutant GFPs, and to a lesser extent the A1 and A5 mutantGFPs, can emit light without the addition of an exogenous substrate orthe use of ultraviolet irradiation. Use of these GFP constructs thusprovides advantages over other visible light reporter vectors whichrequire the use of exogenous substrates, and over other fluorescentreporter vectors which require UV irradiation which may induceundesirable mutations in the host cells.

Example 6 Additional GFP Mutations

To examine the effects of alternative point mutations on GFPfluorescence, mutations are targeted at the tryptophan residue atposition 67 (the-only tryptophan residue in the entire GFP moleculewhich is located in the unique motif Pro-Val-Pro-Trp-Pro (SEQ IDNO:17)). To accomplish this mutation, oligonucleotides are designed tomutate Trp57→His or Trp57→Tyr, in conjunction with the Ser65→Thr mutant(SEQ ID NO:2) or the Phe64→Met; Ser65→Ala mutant (SEQ ID NO:6). Thesemutants are made in the bacterial vector pProEX HTb as described inExample 4, using specific oligonucleotides designed to provide thedesired mutations. The vector constructs are then transfected into hostcells and characterized as above for their fluorescence.

In a similar fashion, mutations are made at other amino acid positionsoutside of the GFP chromophore region. For example, mutations are madeat Arg96, which is probably responsible for stabilizing resonancestructures of the imidazolidone 5-membered ring during ring formationand possibly during excitation, and is therefore a target for more rapidring formation and, hence, faster detection of fluorescence. Mutationsinvolving this residue include Arg96→His.

Mutations are also possible at Phe46, which along with Phe64 separatesthe 5-membered chromophore ring from direct contact with the singletryptophan in the Ser65→Thr GFP (SEQ ID NO:2). By allowing directhydrogen bonding between Trp57 and the ring structure, efficient energytransfer is possible as with the Phe64→Leu; Ser64→Thr mutant. Mutationsinvolving this residue include Phe46→Leu or other hydrophobic residuesthat promote hydrogen bonding.

Mutations are also made at Leu221 and Phe223, which are involved indimer formation. Only three hydrophobic residues are in the dimercontact region; all others are hydrophobic. By mutating Leu221 and/orPhe223 to a hydrophilic or “neutral” residue such as glycine, GFPaggregation, which can be a problem with GFP fusion constructs, may beinhibited.

Mutations are also made at His148, which probably stabilizes thefluorophore and forms hydrogen bonds with Tyr66 and Gln94. Mutations ofHis148 to a residue with a different charge or a different pKa are madeto allow alteration of the excitation and emission spectra of GFP,similar to results seen with Tyr66→His which results in bluefluorescence by GFP.

Finally, mutations introducing a second 5-membered ring structure intothe α-helix of GFP are made, to allow increased fluorescence intensityof the resultant GFP.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1. A nucleic acid molecule encoding a mutant Green Fluorescent Protein,said mutant Green Fluorescent Protein having an amino acid sequencecomprising an amino acid residue lacking an aromatic ring structure atposition 64 and an amino acid residue having a side chain no longer thantwo carbon units in length at position 65, with the provisos that ifsaid residue at position 64 is leucine then said residue at position 65is not cysteine or threonine; if said residue at position 64 is valinethen said residue at position 65 is not alanine; if said residue atposition 64 is methionine then said residue at position 65 is notglycine; and if said residue at position 64 is glycine then said residueat position 65 is not cysteine.